Vaccines with enhanced immune response and methods for their preparation

ABSTRACT

The present invention is concerned with vaccines and their preparation. An effective long-term immune response, especially in mammals, can be produced using a vaccine comprising an antigen encapsulated in liposomes, a suitable adjuvant and a carrier comprising a continuous phase of a hydrophobic substance. The vaccine is particularly effective in eliciting the production of antibodies that recognize epitopes of native proteins.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/246,075 filed Nov. 7, 2000 and U.S. Ser. No. 60/307,159 filedJul. 24, 2001, the disclosures of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to the field of immunology, in particular,to vaccines and their preparation.

BACKGROUND OF THE INVENTION

Generally, vaccines use low doses of a specific antigen to build upresistance in a host to the effects of larger doses of the antigen orsimilar antigenic compounds. Antigens used in vaccines are usually partsof whole organisms or denatured toxins (toxoids) that induce theproduction of antibodies. Unfortunately, only some of the antibodiesproduced bind to the target organism or toxin because, in most cases,the antigen used in the vaccine differs structurally from the target.The limited availability of useful antigens has posed limitations tovaccine development in the past. Advances in genetic engineering havemade the production of antigens by recombinant means possible. However,use of antigens produced by recombinant means often results in poorproduction of antibodies with poor affinity for the target nativeantigen for reasons given above. The effect of immunization can beenhanced when more antibodies with high affinity for their target areproduced. There is a need in the art to develop vaccines that produce anenhanced immune response without increasing the amount of antigen usedin the vaccine. Particularly, there is a need for single administrationvaccines that eliminate or reduce the need for booster immunizations.

Many immunization strategies would benefit from such development.Vaccines that use antigens derived from mammalian, viral, bacterial,fungal or yeast sources have many uses. For example, antigens fromviral, bacterial, fungal or yeast sources are useful in the preventionof disease. Antigens from mammals may be used in cancer therapy orimmunocontraception. Immunocontraceptive vaccines use mammalian derivedantigens that result in transient infertility or sterility of a host,particularly a mammalian host, by favouring the production of antibodieswith affinity for the oocyte surface. Immunocontraceptive vaccines finduse in the control of wild animal populations, including populations offeral domestic animals such as cats.

In particular, feral cat populations have been difficult to control andthreaten many birds and small animals. Stray feral cats also act asvectors for human and animal diseases. Various methods includinghunting, trapping and poisoning have been used in an effort to controlstray cat populations but these methods have met with limited successand with public opposition. Surgical sterilization of feral cats hasbeen increasingly used as a humane tool to lower feral cat populationsduring the last two decades. Acceptance of this procedure is widespread;however, disadvantages include cost, changes in behaviour and risk ofinfection and mortality. Despite the success of large-scale surgicalsterilization, such programs are not financially or logisticallyfeasible in many locations since capture of animals is time-consuming,difficult and stressful for the animal. Immunocontraception offers analternate procedure with lower costs and ease of administration.However, long-term immunocontraception generally requires boostervaccinations, making it impractical for the control of wild andfree-roaming species.

Vaccines generally comprise an antigen, which elicits the immuneresponse in the host, and a variety of carriers, excipients andadjuvants useful for administering the antigen to the host.

Liposomes, which encapsulate the antigen, have increasingly been used invaccine delivery. It has been shown that liposome delivery of denaturedantigens favours the production of antibodies that recognize nativeepitopes (Muttilainen, S., I. Idanpaan-Heikkila, E. Wahlstrom, M.Nurminen, P. H. Makela and M. Sarvas. 1995. “The Neisseria meningitidisouter membrane protein P1 produced in Bacillus subtilis andreconstituted into phospholipid vesicles elicits antibodies to native P1epitopes.” Microbial Pathogen. 18:423-436). While liposomes are usefulvaccine delivery vehicles, their use alone has not provided an effectivesingle dose vaccine, particularly with respect to immunocontraceptivevaccines.

Most immunocontraceptive vaccines use Freund's Complete Adjuvant (FCA)followed by Freund's Incomplete Adjuvant (FIA) in multiple injections toaid production of sufficient antibodies to have an immunocontraceptiveeffect (see Ivanova, et al., 1995. “Contraceptive potential of porcinezona pellucida in cats.” Theriogenology. 43:969-981 and Sacco et al.,1989. “Effect of varying dosage and adjuvants on antibody response insquirrel monkeys (Saimiri sciureus) immunized with the porcine zonapellucida Mr=55,000 glycoprotein (ZP3).” Am. J. Reprod. Immunol.21:1-8). Other adjuvants such as Ribi™ and TiterMax™ have been used bysome investigators. Alum (aluminum phosphate and/or hydroxide) has along history of use as an adjuvant. Alum is the only adjuvant recognizedas safe by the Food and Drug Administration. Many immunocontraceptivevaccines that use alum require a primary injection and several boosterinjections to produce sufficient antibodies for an immunocontraceptiveeffect (see Bagavant et al., 1994. “Antifertility effects of porcinezona pellucida-3 immunization using permissible adjuvants in femalebonnet monkeys (Macaca radiata): reversibility, effect on folliculardevelopment and hormonal profiles.” J. Reprod. Fertil. 102:17-25). Somestudies have shown that alum is not a suitable adjuvant for zonapellucida immunocontraceptive vaccines (see Sacco et al., 1989. Am. J.Reprod. Immunol. 21:1-8 and Bagavant et al., 1994. J. Reprod. Fertil.102:17-25).

Prior art has generally relied on the use of an aqueous medium oroil-in-water emulsions as carriers. For example, Muttilainen et al.(Microbial Pathogen. 18:423-436 (1995) use an aqueous medium incombination with liposomal delivery to elicit an immune response.Popescu (U.S. Pat. No. 5,897,873 issued Apr. 27, 1999 and U.S. Pat. No.6,090,406 issued Jul. 18, 2000), Alving (U.S. Pat. No. 6,093,406 issuedJul. 25, 2000 and U.S. Pat. No. 6,110,492 issued Aug. 29, 2000) andMuderhwa et al. (“Oil-in-water liposomal emulsions: Characterization andpotential use in vaccine delivery”, (December, 1999) J Pharm Sci.88(12):1332-9) also use liposomal systems together with an oil-in-watercarrier as the delivery system in a vaccine. Popescu uses alum withliposomes consisting of cholesterol esterified with succinate or otherorganic acids. U.S. Pat. No. 6,093,406 teaches the use of alum andliposomes comprising Lipid A or non-pyrogenic Lipid A in an oil-in-wateremulsion to deliver a vaccine based on malarial antigens. U.S. Pat. No.6,110,492 and Muderhwa teach the use of liposomes comprising Lipid A ornon-pyrogenic Lipid A in an oil-in-water emulsion to deliver prostratespecific antigens.

Commonly owned U.S. Pat. No. 5,736,141, issued on Apr. 7, 1998, teachesa single dose immunocontraceptive vaccine for seals derived from zonapellucida antigens. While the results achieved with this vaccine aregood, there is still a need for a single-dose, long lastingimmunocontraceptive vaccine effective in a variety of species usingadjuvants approved by the Food and Drug Administration.

There also remains a need for long lasting immunovaccines in generalwhich are effective using a variety of antigens in a variety of speciesusing adjuvants approved by the Food and Drug Administration.

SUMMARY OF THE INVENTION

In accordance with the invention, there is provided a composition foruse as a vaccine, comprising:

-   -   (a) a carrier comprising a continuous phase of a hydrophobic        substance;    -   (b) liposomes;    -   (c) an antigen; and,    -   (d) a suitable adjuvant.

There is further provided a method for potentiating an immune responsein an animal, which method comprises administering to the animal aneffective amount of a vaccine composition comprising:

-   -   (a) a carrier comprising a continuous phase of a hydrophobic        substance;    -   (b) liposomes;    -   (c) an antigen; and,    -   (d) a suitable adjuvant.

Still further there is provided a method of preparing a vaccinecomposition comprising the steps of:

-   -   (a) encapsulating an antigen or an antigen/adjuvant complex in        liposomes to form liposome-encapsulated antigen;    -   (b) mixing the liposome-encapsulated antigen with a carrier        comprising a continuous phase of a hydrophobic substance; and,    -   (c) adding a suitable adjuvant if an antigen/adjuvant complex is        not used in part (a).

Unexpectedly and uniquely, it has now been found that using a continuousphase of a hydrophobic substance as the carrier in a vaccine compositionof the present invention enhances the immune response. The enhancedresponse is characterized by long-lived high antibody titres following asingle vaccine administration resulting in enhanced duration of theimmune response. This is particularly true for vaccines that alsocomprise liposome-encapsulated antigen and an adjuvant (or mixture ofantigen/adjuvant). Vaccine compositions of the present invention aregenerally effective as a single dose providing a long-term immuneresponse in a variety of species, typically not requiring boosters.

DETAILED DESCRIPTION OF THE INVENTION

While not being held to any particular theory of action, it is thoughtthat, when a vaccine composition of the present invention is used, IgGantibody production occurs in two phases and the antibodies produced ineach phase differ in their epitope recognition. The antibodies producedin the second phase of IgG production have more affinity for nativeprotein antigens, thus making the vaccine more effective. Use ofconventional vaccines with a primary and booster injection producesantibodies having different binding specificity for an antigen than useof a vaccine composition of the present invention.

The carrier comprises a continuous phase of a hydrophobic substance,preferably a liquid hydrophobic substance. The continuous phase may bean essentially pure hydrophobic substance, a mixture of hydrophobicsubstances, an emulsion of water-in-a hydrophobic substance or anemulsion of water-in-a mixture of hydrophobic substances.

Hydrophobic substances that are useful in the present invention arethose that are pharmaceutically and/or immunologically acceptable.Ideally, the hydrophobic substance is one that has been approved for useby health regulatory agencies such as the U.S. Food and DrugAdministration. The carrier is preferably a liquid but certainhydrophobic substances that are not liquids at atmospheric temperaturemay be liquified, for example by warming, and are also useful in thisinvention.

Oil or water-in-oil emulsions are particularly suitable carriers for usein the present invention. Oils should be pharmaceutically and/orimmunologically acceptable. Preferred examples of oils are mineral oil(especially light or low viscosity mineral oil), vegetable oil (e.g.corn or canola oil), nut oil (e.g. peanut oil) and squalene. A lowviscosity mineral oil is most preferred. Animal fats and artificialhydrophobic polymeric materials, particularly those that are liquid atatmospheric temperature or that can be liquified relatively easily, mayalso be used.

The amount of hydrophobic substance used is not critical but istypically from about 0.1 ml per dose to about 1.5 ml per dose, dependingon the size of the animal and the amount of antigen being used. Forsmall animals, the amount of hydrophobic substance is preferably fromabout 0.20 ml to about 1.0 ml per dose, while for large animals, theamount is preferably from about 0.45 ml to about 1.5 ml per dose.Typically, 0.25 ml per dose is used for small animals while 0.5 ml perdose is used for large animals.

Suitable antigens are any chemicals that are capable of producing animmune response in a host organism. Preferably, the antigen is asuitable native, non-native, recombinant or denatured protein orpeptide, or a fragment thereof, that is capable of producing the desiredimmune response in a host organism. Host organisms are preferablyanimals (including mammals), more preferably cats, rabbits, horsesand/or deer. The antigen can be of a viral, bacterial, protozoal ormammalian origin. Antigens are generally known to be any chemicals(typically proteins or other peptides) that are capable of eliciting animmune response in a host organism. More particularly, when an antigenis introduced into a host organism, it binds to an antibody on B cellscausing the host to produce more of the antibody. For a generaldiscussion of antigens and the immune response, see Kuby, J., Immunology3^(rd) Ed. W.H. Freeman & C. NY (1997), the disclosure of which ishereby incorporated by reference.

Antigens that elicit an immune response related to cancer, contraceptionand other biological conditions or effects may be used in thepreparation of immunovaccines. Some typical, non-limiting examples ofantigens that may be used are alcohol dehydrogenase (ADH),streptokinase, hepatitis B surface antigen and zona pellucida (ZP)glycoproteins.

When the desired immune response is contraception in mammals, the targetepitopes are found on mammalian oocytes. Zona pellucida (ZP)glycoproteins or recombinant proteins or peptide fragments derivedtherefrom may be used in this case. In particular, heat extractedsolubilized isolated zona pellucida glycoproteins (SIZP) may be used asthe antigen in an immunocontraceptive vaccine. More particularly,soluble intact porcine zona pellucida may be used.

The amount of antigen used in a dose of the vaccine composition can varydepending on the type of antigen and the size of the host. One skilledin the art will be able, to determine, without undue experimentation,the effective amount of antigen to use in a particular application.

In the case of SIZP, the amount typically used falls in the range fromabout 15 μg to about 2 mg per dose. Preferably, the range is from about20 μg to about 2 mg per dose, more preferably from about 20 μg to about200 μg, and even more preferably from about 40 μg to about 120 μg.Typically, the amount for a small animal is about 50 μg per dose whilefor a large animal it is about 100 μg per dose.

In compositions of the present invention, antigens produce enhancedlevels of host antibodies that bind to native epitopes of the targetprotein. This is the case even though the antigen may be a non-native,recombinant or denatured protein or peptide, or a fragment thereof.While not wishing to be held, to any particular theory, this may be dueto the antigen being held in a native-like three-dimensionalconformation in the liposomes.

Liposomes are completely closed lipid bilayer membranes containing anentrapped aqueous volume. Liposomes may be unilamellar vesicles(possessing a single bilayer membrane) or multilamellar vesicles(onion-like structures characterized by multimembrane bilayers, eachseparated from the next by an aqueous layer. A general discussion ofliposomes can be found in Gregoriadis G. (1990) Immunological adjuvants:A role for liposomes, Immunol. Today 11:89-97 and Frezard, F. (1999)Liposomes: From biophysics to the design of peptide vaccines. Braz. J.Med. Bio. Res 32:181-189, the disclosures of which are herebyincorporated by reference.

Although any liposomes may be used in this invention, includingliposomes made from archaebacterial lipids, particularly usefulliposomes use phospholipids and unesterified cholesterol in the liposomeformulation. The cholesterol is used to stabilize the liposomes and anyother compound that stabilizes liposomes may replace the cholesterol.Other liposome stabilizing compounds are known to those skilled in theart. The use of the particularly preferred liposomes may result inlimiting the electrostatic association between the antigen and theliposomes. Consequently, most of the antigen may be sequestered in theinterior of the liposomes.

Phospholipids that are preferably used in the preparation of liposomesare those with at least one head group selected from the groupconsisting of phosphoglycerol, phosphoethanolamine, phosphoserine,phosphocholine and phosphoinositol. More preferred are liposomes thatcomprise lipids in phospholipon 90 G.

The amount of lipid used to form liposomes depends on the antigen beingused but is typically in a range from about 0.05 gram to about 0.5 gramper dose of vaccine. Preferably, the amount is about 0.1 gram per dose.When unesterified cholesterol is also used in liposome formulation, thecholesterol is used in an amount equivalent to about 10% of the amountof lipid. The preferred amount of cholesterol is about 0.01 gram perdose of vaccine. If a compound other than cholesterol is used tostabilize the liposomes, one skilled in the art can readily determinethe amount needed in the formulation.

In a more preferred aspect, the vaccine compositions of the presentinvention are essentially free from Lipid A, including non-pyrogenicLipid A. For the purposes of this specification, when the term Lipid Ais used, it is understood to encompass non-pyrogenic Lipid A as well.Lipid A is often found in liposomal formulations of the prior art. LipidA has many undesirable side-effects which may be overcome usingnon-pyrogenic Lipid A, but even then, Lipid A has many pharmaceuticalreactions other than the pyrogenic one and may still cause many adversereactions. It is therefore desirable to exclude Lipid A from thecompositions of this invention.

Suitable adjuvants are alum, other compounds of aluminum, Bacillus ofCalmette and Guerin (BCG), TiterMax™, Ribi™, Freund's Complete Adjuvant(FCA) and a new adjuvant disclosed by the United States Department ofAgriculture's (USDA) National Wildlife Research Center on their web siteat http://www.aphis.usda.gov/ws/nwrc/pzp.htm based on Johne's antigen.Alum, other compounds of aluminum, TiterMax™ and the new USDA adjuvantare preferred. Enhanced immune response is found even when the adjuvantis alum, which is surprising in view of the prior art (Sacco et al.1989. Am. J. Reprod. Immunol., 21:1-8). Alum is particularly preferredas the adjuvant.

Alum is generally considered to be any salt of aluminum, in particular,the salts of inorganic acids. Hydroxide and phosphate salts areparticularly useful as adjuvants. A suitable alum adjuvant is sold underthe trade name, ImjectAlum™ (Pierce Chemical Company) that consists ofan aqueous solution of aluminum hydroxide (45 mg/ml) and magnesiumhydroxide (40 mg/ml) plus inactive stabilizers. Alum is a particularlyadvantageous adjuvant since it already has regulatory approval and it iswidely accepted in the art.

The amount of adjuvant used depends on the amount of antigen and on thetype of adjuvant. One skilled in the art can readily determine theamount of adjuvant needed in a particular application. Forimmunocontraception, a suitable quantity of ImjectAlum™ for a rabbit is0.1 ml/dose of vaccine, whereas, a suitable quantity of ImjectAlum™ fora horse is 0.5 ml/dose.

The vaccine composition is generally formulated by: encapsulating anantigen or an antigen/adjuvant complex in liposomes to formliposome-encapsulated antigen and mixing the liposome-encapsulatedantigen with a carrier comprising a continuous phase of a hydrophobicsubstance. If an antigen/adjuvant complex is not used in the first step,a suitable adjuvant may be added to the liposome-encapsulated antigen,to the mixture of liposome-encapsulated antigen and carrier, or to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen. The order of the process may depend on the type of adjuvantused. Typically, when an adjuvant like alum is used, the adjuvant andthe antigen are mixed first to form an antigen/adjuvant complex followedby encapsulation of the antigen/adjuvant complex with liposomes. Theresulting liposome-encapsulated antigen is then mixed with the carrier.(It should be noted that the term “liposome-encapsulated antigen” mayrefer to encapsulation of the antigen alone or to the encapsulation ofthe antigen/adjuvant complex depending on the context.) This promotesintimate contact between the adjuvant and the antigen and may, at leastin part, account for the surprisingly good immune response when alum isused as the adjuvant. When another is used, the antigen may be firstencapsulated in liposomes and the resulting liposome-encapsulatedantigen is then mixed into the adjuvant in a hydrophobic substance.

In formulating a vaccine composition that is substantially free ofwater, antigen or antigen/adjuvant complex is encapsulated withliposomes and mixed with a hydrophobic substance. In formulating avaccine in an emulsion of water-in-a hydrophobic substance, the antigenor antigen/adjuvant complex is encapsulated with liposomes in an aqueousmedium followed by the mixing of the aqueous medium with a hydrophobicsubstance. In the case of the emulsion, to maintain the hydrophobicsubstance in the continuous phase, the aqueous medium containing theliposomes may be added in aliquots with mixing to the hydrophobicsubstance.

In all methods of formulation, the liposome-encapsulated antigen may befreeze-dried before being mixed with the hydrophobic substance or withthe aqueous medium as the case may be. In some instances, anantigen/adjuvant complex may be encapsulated by liposomes followed byfreeze-drying. In other instances, the antigen may be encapsulated byliposomes followed by the addition of adjuvant then freeze-drying toform a freeze-dried liposome-encapsulated antigen with externaladjuvant. In yet another instance, the antigen may be encapsulated byliposomes followed by freeze-drying before the addition of adjuvant.Freeze-drying may promote better interaction between the adjuvant andthe antigen resulting in a more efficacious vaccine.

Formulation of the liposome-encapsulated antigen into a hydrophobicsubstance may also involve the use of an emulsifier to promote more evendistribution of the liposomes in the hydrophobic substance. Typicalemulsifiers are well-known in the art and include mannide oleate(Arlacel™ A), lecithin, Tween™ 80, Spans™ 20, 80, 83 and 85. Mannideoleate is a preferred emulsifier. The emulsifier is used in an amounteffective to promote even distribution of the liposomes. Typically, thevolume ratio (v/v) of hydrophobic substance to emulsifier is in therange of about 5:1 to about 15:1 with a ratio of about 10:1 beingpreferred.

Administration of the vaccine composition can be done by any convenientmethod and will depend on the antigen being used. Vaccine compositionsmay be administered parenterally (including intramuscularly,sub-cutaneously) or rectally. Parenteral administration is preferred.

For parenteral application, particularly convenient unit dosage formsare ampoules. Techniques that deliver the vaccine by injection and byremote delivery using darts, spring loaded syringes with jab sticks,air/carbon dioxide powered rifles, Wester gun and/or Ballistivet™biobullets and retain the biological activity are particularlypreferred.

The amount of vaccine composition administered to a host may depend onthe amount of antigen used in a dose and on the effective amount ofantigen required for a particular application. In the case of SIZP, thesize of each dose administered to an animal is typically from about 0.25ml to about 2.0 ml depending on the size of the animal. For smalleranimals (for example, cats, rabbits, etc.) the size of the dose istypically about 0.5 ml while for larger animals (for example, horses,fallow-deer, white-tail deer, etc.) the size of the dose is typicallyabout 1.0 ml. Typically, even when the amount of SIZP is varied, thedose size is kept fairly constant.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described by way of non-limiting exampleshaving regard to the appended drawing in which:

FIG. 1 is a diagram showing the position of recombinant ZPB1 and ZPB2;ZPC1 and ZPC2 of ZPB and ZPC proteins of porcine zona pellucida thatwere generated in the pRSET vectors.

EXAMPLES Example 1 Preparation of the Vaccine Composition

The vaccine composition can be formulated to be water-free or to containvarious quantities of water (by using an aqueous medium, for example,saline, phosphate buffered saline (PBS) or pyrogen-free water) whilemaintaining a continuous oil phase. Procedure 1 described below appliesto the water-free formulation of the vaccine composition. Procedure 2described below applies to the water containing formulation of thevaccine composition, that is, the water-in-oil emulsion. The twoprocedures can also vary depending on the adjuvant being used. Asexamples, method A applies to formulations of the vaccine compositioncontaining alum and method B applies to formulations containing Freund'sComplete Adjuvant (FCA). Other adjuvants may be accommodated by adaptingeither method A or method B. The procedures described below incorporateporcine soluble intact zona pellucida (SIZP) as antigen, other antigenscan replace SIZP in the formulation. For example, alcohol dehydrogenase(ADH), streptokinase or hepatitis B surface antigen can also be used asthe antigen.

Procedure 1: Water-Free Formulation.

Method A. Alum adjuvant.

SIZP is prepared as previously described (Brown, R. G., W. D. Bowen, J.D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B. Pohajdak.(1997) Temporal trends in antibody production in captive grey seals,harp and hooded seals to a single administration immunocontraceptivevaccine. J. Reproductive Immunology 35:53-64). The quantity of SIZPneeded for the number of doses of the vaccine being prepared is weighed(the usual quantity of SIZP used for immunization is 50 μg for smallanimals and 100 μg for large animals). The SIZP is dissolved inpyrogen-free distilled water to give a final concentration of 2 mg/ml.An equal volume of ImjectAlum™ (an alum product from Pierce ChemicalCo., catalogue #77161) is added and the suspension is mixed, thenfreeze-dried.

To form liposomes, phospholipon 90 G (or other lipids selected fromphosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine,phosphoinositol, archaebacterial lipids, without limitation, that form aclosed lipid bilayer containing an entrapped antigen) is weighed (0.1g/dose of the vaccine composition). The phospholipon 90 G is mixed withcholesterol (0.01 g/dose of vaccine composition) and the mixture isdissolved in chloroform:methanol (1/1; v/v; 1.5 ml/dose of the vaccinecomposition). Cholesterol can be replaced with other compounds thatstabilize liposomes at concentrations determined by those skilled in theart. Washed glass beads (approximately 3 mm in diameter; 15 ml for 10doses of the vaccine) are added and the mixture is evaporated underreduced pressure using a rotary evaporator until free ofchloroform:methanol. To ensure removal of all chloroform:methanol, themixture is placed in a dessicator under reduced pressure overnight atroom temperature.

The freeze-dried SIZP/alum complex is suspended in pyrogen-freedistilled water (5 ml/mg SIZP) and the suspension added to the flaskcontaining the mixture of phospholipon 90 G/cholesterol coating theflask and glass beads. The contents of the flask are allowed to standwithout agitation for 30 minutes. After 30 minutes, the flask is placedin a water bath at 35-40° C. and stirred gently with a spatula to formthe liposomes. A microscope is used to evaluate liposome formation andstirring is continued with increased shaking until the mixture containspredominately multilamellar liposomes recognized by those skilled in theart. The liposomes are freeze-dried and the resulting freeze-driedliposomes are suspended in low viscosity mineral oil (0.25 ml oil/dosefor small animals and 0.5 ml oil/dose for large animals) containingmannide oleate as an emulsifier (10:1:oil:emulsifier:v/v). Sinceliposomes are suspended in oil and are not in solution, it is necessaryto determine if the procedures used result in an even distribution ofSIZP in each dose. To determine if freeze-dried liposomes containingSIZP are equally distributed in oil, SIZP is labelled with ¹⁴C byreductive methylation (Jentoft, N. and D. G. Dearborn. 1979. Labellingof proteins by reductive methylation using sodium cyanoborohydride. J.Biol. Chem. 254:4359-4365, the disclosure of which is herebyincorporated by reference) and the radioactive SIZP used to prepare twopreparations of the vaccine. Individual doses of the vaccine areprepared and radioactivity in each dose determined, thereby determiningthe content of SIZP in each dose of the vaccine (Table 1). Thedistribution of SIZE, in each dose of the vaccine is highly reproducible(standard deviation, SD, was less than +/−10%).

TABLE 1 Distribution of ¹⁴C-labelled SIZP in doses of the vaccine fromtwo preparations Preparation 1 Preparation 2 Sample No. μg SIZP/dose μgSIZP/dose 1 68 55 2 66 53 3 57 53 4 65 62 5 56 59 6 51 57 7 62 58 8 5560 9 69 51 10 54 59 11 52 57 12 61 61 13 64 61 14 61 60 15 — 56 Average60 57 Standard Deviation 5.9 3.2

Method B. FCA Adjuvant.

Preparation of the vaccine composition to contain FCA as adjuvant inplace of alum, is similar to method A, except SIZP by itself, ratherthan as a SIZP/alum complex, is encapsulated in liposomes as describedabove. The liposomes containing SIZP are freeze-dried, and thefreeze-dried liposomes are added to FCA in aliquots with mixing topromote an equal distribution of liposomes in the oil. The resultingsuspension of freeze-dried liposomes containing SIZP in FCA isadministered to animals being vaccinated.

Procedure 2. Water-Containing Formulation. Method A. Alum Adjuvant.

SIZP is prepared as previously described (Brown, R. G., W. D. Bowen, J.D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B. Pohajdak.(1997) Temporal trends in antibody production in captive grey seals,harp and hooded seals to a single administration immunocontraceptivevaccine. J. Reproductive Immunology 35:53-64, the disclosure of which ishereby incorporated by reference). The quantity of SIZP needed for thenumber of doses of the vaccine being prepared is weighed (the usualquantity of SIZP used for immunization is 50 μg for small animals and100 μg for large animals). The SIZP is dissolved in pyrogen-freedistilled water to give a final concentration of 2 mg/ml. An equalvolume of ImjectAlum™ (an alum product from Pierce Chemical Co.,catalogue #77161) is added and the suspension is mixed, thenfreeze-dried.

To form liposomes, phospholipon 90 G (or other lipids selected fromphosphoglycerol, phosphoethanolamine, phosphoserine, phosphocholine,phosphoinositol, etc. that form a closed lipid bilayer containing anentrapped aqueous volume) is weighed (0.1 g/dose of the vaccinecomposition). The phospholipon 90 G is mixed with cholesterol (0.01g/dose of the vaccine composition) and the mixture is dissolved inchloroform:methanol (1/1; v/v; 1.5 ml/dose of the vaccine composition).Cholesterol can be replaced with other compounds that stabilizeliposomes at concentrations determined by those skilled in the art.Washed glass beads (approximately 3 mm in diameter; 15 ml for 10 dosesof the vaccine) are added and the mixture is evaporated under reducedpressure using a rotary evaporator until free of chloroform:methanol. Toensure removal of all chloroform:methanol, the mixture is placed in adessicator under reduced pressure overnight at room temperature.

The freeze-dried SIZP/alum complex is suspended in saline (5 ml/mg SIZP)and the suspension is added to the flask containing the mixture ofphospholipon 90 G/cholesterol coating the flask and glass beads. Thecontents of the flask are allowed to stand without agitation for 30minutes. After 30 minutes, the flask is placed in a water bath at 35-40°C. and stirred gently with a spatula to form the liposomes. A microscopeis used to evaluate liposome formation and stirring is continued withincreased shaking until the mixture contains predominately multilamellarliposomes recognized by those skilled in the art. The aqueous suspensionof liposomes (0.25 ml/dose for small animals and 0.5 ml/dose for largeanimals) is added to low viscosity mineral oil (0.25 ml oil/dose forsmall animals and 0.5 ml oil/dose for large animals) containing mannideoleate as an emulsifier (10:1:oil:emulsifier:v/v). The aqueoussuspension of liposomes is added to the low mineral oil phase inaliquots with mixing between aliquots to maintain the continuous oilphase.

Method B. FCA Adjuvant.

Preparation of the vaccine composition to contain FCA as adjuvant inplace of alum, is similar to method A, except SIZP by itself, ratherthan as a SIZP/alum complex, is encapsulated in liposomes as describedabove. The aqueous suspension of liposomes is added to FCA in aliquotswith mixing between aliquots to maintain the continuous oil phase. Theresulting aqueous suspension of liposomes containing SIZP in FCA isadministered to animals being vaccinated.

Note: In some trials, the quantity of SIZP is varied to study theresponse of immunized animals to different quantities of antigen. Insuch experiments, the volume of the vaccine administered to smallanimals (cats, rabbits, etc.) was 0.5 ml and the volume administered tolarge animals (horses, fallow deer, white-tailed deer, etc.) was 1.0 ml.In such cases, the quantity of liposomes in each dose of the vaccine ismaintained constant while the quantity of antigen encapsulated inliposomes varied.

Example 2 Immunization of Rabbits Against Native and Denatured YeastAlcohol Dehydrogenase (ADH)

The vaccine composition is unique in producing high titers of anti-SIZPantibodies that are long-lasting following a single administration. Todetermine if the vaccine composition would produce high antibody titerswith other antigens, particularly proteins that are not bound to cellmembranes, rabbits are immunized with yeast alcohol dehydrogenase (ADH).Two forms of ADH are used as antigen, namely, native ADH and ADH thathad been treated to denature the protein. To denature ADH, ADH istreated with mercaptoethanol (10% v/v in Tris buffer, 0.1 M, pH 7.5, 30min, 100° C.). The solution is dialyzed against distilled water andfreeze-dried. Four rabbits (2 for each treatment) are immunized withnative or denatured ADH (40 μg) using a primary injection with Freund'scomplete adjuvant (FCA) followed by a booster injection with Freund'sincomplete adjuvant (FIA) given one month later. The post-immunizationperiod is considered to have begun after the booster injection. At thesame time as the booster injection is administered, four rabbits (2 foreach treatment) are immunized by single administration with eithernative or denatured ADH (40 μg) using the vaccine composition, that isADH is encapsulated in liposomes that are suspended in saline (0.5 ml)and emulsified in FCA (0.5 ml). Anti-ADH titers are measured by ELISAusing both native and denatured ADH (Table 2).

When rabbits are immunized with native ADH, the resulting serumcontained similar quantities of anti-ADH antibodies when native ADH isdelivered with the vaccine composition or by using a primary injectionwith one booster. In contrast, serum from rabbits immunized withdenatured ADH delivered with the vaccine composition contain 2.7 timesmore antibody that bound to native ADH than serum from rabbits that areimmunized with denatured ADH with a primary injection and one booster(P<0.01; T=4.14; df=6). In all cases, titers are higher in rabbitsimmunized with native ADH than when rabbits were immunized withdenatured ADH. This indicates that native ADH is a better antigen thandenatured ADH. Since many protein antigens are denatured to some degreeduring extraction and isolation or when produced by recombinant means,increased production of antibodies that bind better to native proteinscan significantly improve the outcome of vaccination as demonstrated byimmunocontraception of a variety of mammals with SIZP using the vaccinecomposition of the present invention.

Furthermore, anti-ADH sera from rabbits 237 and 238 recognize denaturedADH in Western blots with about 4-5 times the intensity of anti-ADH serafrom rabbits 235 and 236. This confirms the results of titermeasurements indicating that immunization of rabbits with the vaccinecomposition favours the production of anti-ADH antibodies that bindbetter to native ADH since many proteins are known to refold during theWestern protocol to a more native state. This conclusion is supported byMuttilainen et al. (1995) in a study of Neisseria meningitidis outermembrane protein P1, who found antibodies to native P1 were elicited inmice vaccinated with denatured P1 constituted into phospholipid vesicles(liposomes). However, Muttilainen et al. (1995) did not use oil in theirvaccine formulation, therefore, their immunization protocol wasdifferent than the present invention.

TABLE 2 Production of anti-ADH antibodies by rabbits immunized withnative or denatured ADH delivered with and without liposomeencapsulation Anti-ADH titer Immunization (% of reference serum)¹ RabbitNative ADH³ Denatured ADH³ ID No. Antigen Delivery² 4⁴ 5⁴ 4⁴ 5⁴ 231native −Liposomes 187 148 19 23 ADH 232 native −Liposomes 200 161 17 15ADH 233 native +Liposomes 239 156 21 14 ADH 234 native +Liposomes 100100 19 11 ADH 235 denatured −Liposomes 41 37 7 3 ADH 236 denatured−Liposomes 30 18 7 4 ADH 237 denatured +Liposomes 101 71 8 3 ADH 238denatured +Liposomes 101 63 12 7 ADH ¹Anti-ADH serum from rabbit 234 isused as the reference serum. ²Native and denatured ADH are administeredwith the vaccine composition, that is encapsulated in liposomes with FCAas a single i.m. injection (+liposomes) or suspended in FCA followed onemonth later as a booster injection using FIA (−liposomes). Rabbitsreceive 40 μg ADH with each administration. ³ADH is purchased from SigmaChemical Co. Denatured ADH is produced by treating native ADH withmercaptoethanol and heating to 100° C. for 30 minutes. The resultingdenatured ADH contain three major proteins having molecular weights of26, 33 and 40 kDa. Titers are measured by ELISA using native anddenatured ADH as antigen to coat ELISA plates in separatedeterminations. ⁴Post-immunization in months. Note: The reference serumnoted in Table 2 is rabbit serum ID No. 234.

Example 3 Immunocontraception of Rabbits

Sera from rabbits immunized with a placebo vaccine that contained allingredients of the vaccine composition except the antigen (porcine SIZP)contain no anti-porcine SIZP antibodies (see Table 3A). Immunization ofrabbits with porcine SIZP (40 μg) encapsulated in liposomes containingphosholipon 90 G (0.1 g), cholesterol (0.01 g) in saline (0.5 ml)emulsified in FCA adjuvant (0.5 ml) produce high titers of anti-SIZPantibodies during the 12 month post-immunization monitoring periodfollowing a single administration of the vaccine. Immunization ofrabbits with porcine SIZP (40 μg) encapsulated in liposomes with MF 59adjuvant (0.5 ml) produce low anti-SIZP titers. In contrast,immunization of rabbits with porcine SIZP encapsulated in liposomes withalum adjuvant (100 μl, Pierce ImjectAlum™) produce anti-porcine SIZPtiters that are less than titers produced using FCA in earlypost-immunization but the titers are less different than between thealum and FCA runs by the 12^(th) month of post-immunization. Breeding ofrabbits established that a single administration of the vaccine usingFCA or alum reduces fertility of rabbits by 79 and 74% respectively(Table 3B). Immunization of rabbits by a single injection of SIZP (40μg) that is not encapsulated in liposomes with Gerbu adjuvant produceslow anti-porcine SIZP titers (Table 3A). As expected based on anti-SIZPtiters, rabbits immunized with SIZP that are not encapsulated inliposomes with Gerbu adjuvant have the same fertility as rabbits thatreceive the placebo vaccine (Table 3B). These results indicate thatvaccines comprising liposome-encapsulated antigen produce good resultsand that FCA and alum, particularly alum, are especially good adjuvants.

TABLE 3A Effect of adjuvants on the production of anti-porcine SIZPantibodies by rabbits¹ Post-immunization anti-porcine SIZP titer (% ofreference ID Time (months) No. 0 1 2 3 4 5 6 7 11 12 Placebo 2 0 1 0 0 00 0 0 0 0 13 0 0 0 0 0 0 0 0 0 0 14 0 0 0 0 0 0 0 0 0 0 FCA 3 0 145 11294 82 45 23 24 20 20 15 0 100 71 68 83 19 29 26 18 25 16 0 125 111 122128 111 74 92 75 63 MF 59 1 0 3 1 1 1 2 0 ND ND ND 7 0 10 9 3 3 5 2 NDND ND 19 0 12 6 3 3 3 3 ND ND ND 20 0 37 21 13 15 13 10 ND ND ND Alum 110 19 12 12 11 14 18 7 8 10 12 0 20 10 10 8 6 11 5 2 4 23 0 31 16 40 2220 33 28 37 28 24 0 36 21 40 27 24 40 36 34 34 Gerbu without liposomeencapsulation of SIZP 9 0 5 2 1 1 1 1 1 ND ND 10 0 11 3 3 2 2 3 1 ND ND21 0 17 4 6 19 5 6 2 ND ND 22 0 24 3 6 3 4 4 7 ND ND ¹Rabbits receive asingle administration of the placebo vaccine, vaccine that containedporcine SIZP (40 μg) encapsulated in liposomes (0.25 ml) with eitherFCA, MF 59 or alum adjuvants (0.25 ml) or vaccine that contained porcineSIZP (40 μg) dissolved in saline (0.25 ml) with gerbu adjuvant (0.25ml). ND = not determined.

The reference serum used is from a rabbit immunized with porcine zonapellucida using a primary injection with Freund's complete adjuvant and2 booster injections with Freund's incomplete adjuvant.

TABLE 3B Effect of adjuvants on the fertility of rabbits immunizedagainst porcine SIZP Live births/mating¹ Average live % reduction in IDNo 1 2 3 births/mating fertility Placebo 2 6 0 4 5.1 0 13 7 1 10 14 6 75 FCA 3 0 0 0 1.1 79 15 0 8 5 16 0 NM 0 MF 59 1 11 NM 10 6.1 0 7 0 0 NM19 5 11 11 20 6 0 7 Alum 11 0 0 6 1.3 74 12 0 0 0 23 0 0 0 24 3 5 2Gerbu without liposome encapsulation 9 0 0 11 5.3 0 10 0 0 0 21 8 9 1122 10 7 11 NM = paired repeatedly with males without a successful mating¹Live births following a successful mating. The mating intervals were 65± 10, 141 ± 14 and 216 ± 14 days post-immunization for matings 1, 2 and3 respectively.

Example 4 Immunocontraception in Cats

Twenty-nine specific pathogen free domestic short hair cats are housedat the specific pathogen free facility at the University of Floridaunder the supervision of Dr. Julie Levy and Mr. Shawn Gorman. Estruscycling is monitored by daily observation and vaginal cytology. Vaccinecompositions of the present invention, placebo vaccines and serumsamples are coded as part of a double-blind study. The cats are dividedrandomly into 3 groups of nine or ten cats each. One group receives aplacebo vaccine that contains all components of the vaccine compositionexcept the antigen (porcine SIZP) by intramuscular injection. Each catin this group receives liposomes containing no antigen in saline (0.25ml) suspended in FCA (0.25 ml). Each cat in a second group of nine catsis immunized by intramuscular injection with the vaccine compositioncontaining SIZP (135 μg) encapsulated in liposomes in saline (0.25 ml)and suspended in FCA (0.25 ml). Each cat in a third group of nine catsis immunized by intramuscular injection with the vaccine compositioncontaining porcine SIZP=20 (200 n) with alum (0.12 ml, Pierce ChemicalCo., catalogue number 77161) encapsulated in liposomes in saline (0.12ml) and suspended in a suitable pharmacological carrier. Production ofanti-SIZP antibodies in cats is measured by ELISA using proteinA/alkaline phosphatase (Brown, R. G., W. D. Bowen, J. D. Eddington, W.C. Kimmins, M. Mezei, J. L. Parsons, B. Pohajdak. (1997) Temporal trendsin antibody production in captive grey seals, harp and hooded seals to asingle administration immunocontraceptive vaccine. J. ReproductiveImmunology 35:53-64).

A single administration of the vaccine composition using FCA producesanti-SIZP antibody titers that reached maximal titers within 2 months(Table 4). The average two months post-immunization titer is 58±2% ofthe reference serum which decreased to 41±4% of the reference serum atfour months post-immunization when a proven male cat is introduced tothe colony. Cats that receive a single administration of the vaccinecomposition using alum as adjuvant produce anti-SIZP antibodies with anaverage titer of 67±2% of the reference serum two monthspost-immunization.

Monthly serum samples from cats that are immunized with the placebovaccine containing all components of the vaccine composition except theantigen, have an average anti-SIZP titer of 0.6±0.2% of the referenceserum during the post-immunization monitoring period. Therefore, it isapparent that cats that received the placebo vaccine will producekittens during the post-immunization period.

TABLE 4 Production of anti-SIZP antibodies by cats immunized with thevaccine composition of the invention Anti-SIZP titer (% of referenceserum) Cat ID Post-immunization (months) No. 0 1 2 3 4 5 6 7 8 9 10 11Placebo 3 0 0 0 0 1 0 0 0 0 7 4 2 4 0 0 0 0 2 0 0 3 1 2 2 0 8 0 3 0 1 00 0 1 1 2 2 0 15  1 0 1 0 0 0 0 2 1 ND ND ND A 0 0 3 0 1 0 0 0 0 2 2 0 B0 0 0 1 1 1 0 ND 2 2 0 0 E 0 1 1 1 0 0 0 2 5 ND ND ND F 0 1 1 0 0 0 0 20 ND ND ND I 0 1 0 1 1 0 0 0 2 0 0 ND N 0 1 0 0 1 1 1 2 1 ND ND NDVaccine with FCA 1 0 60 57 68 46 56 23 46 15 40 23 23 2 0 60 53 58 44 2523 69 46 68 70 62 9 0 47 56 41 56 20 58 78 103 74 86 ND 13  0 42 64 5366 55 47 29 33 12 16 ND 14  0 48 51 30 45 40 10 20 9 10 12 7 C 0 56 5449 34 21 16 12 14 24 14 16 D 0 58 60 67 59 52 32 19 48 ND ND ND L 0 4761 40 28 16 46 37 59 70 59 72 G 0 59 65 79 85 81 92 94 96 115 152 79 O 048 42 53 42 24 26 28 25 31 16 10 Vaccine with alum 1P 1 60 60 70 46 4725 18 41 26 6 ND 1S 0 73 56 43 24 42 19 18 18 14 4 ND 1T 0 62 62 58 3034 14 12 8 11 4 ND 1V 2 70 68 60 40 36 12 12 ND ND ND ND 1Y 1 84 82 8481 80 67 34 44 28 ND ND 1Z 0 77 75 71 54 72 54 26 35 21 9 ND Z1 0 61 7495 83 100 98 92 70 42 ND ND Z2 0 77 79 63 34 50 41 28 18 11 ND ND Z3 065 72 61 55 30 35 18 20 6 ND ND Z4 0 73 ND 68 53 29 32 17 12 13 ND ND ND= not determined

Example 5 Immunocontraception of Deer

Forty-one fallow deer (Dama dama) does on James Island, a 360-hectareisland that lies off the coast of southern British Columbia, areimmunized with the vaccine composition using FCA as adjuvant. Anothergroup of forty fallow deer does are immunized with the vaccinecomposition using alum as the adjuvant. For capture, the deer are baitedinto a large (200×200 meter) pen that is connected to a series of fencedenclosures and a raceway that terminates in a small building. Beforeimmunization, each deer is physically restrained and given a numberedear tag, a colored plastic collar or radio collar with a mortalitysensor, and a PIT (permanent identification transponder) tag bearing aunique code. Thus, if a treated deer loses all external marks, it couldstill be recognized as a treated animal from injury resulting from lossof ear tag and as a particular deer from the PIT tag. Each captive doeis injected intramuscularly in the rump with SIZP (100 μg) encapsulatedin liposomes with FCA or alum adjuvants. Untreated does serve ascontrols.

Anti-SIZP titers are measured as previously described (Brown, R. G., W.D. Bowen, J. D. Eddington, W. C. Kimmins, M. Mezei, J. L. Parsons, B.Pohjajdak. (1997) Temporal trends in antibody production in captive greyseals, harp and hooded seals to a single administrationimmunocontraceptive vaccine. J. Reproductive Immunology 35:53-64) exceptthat protein G/alkaline phosphatase replaced protein A/alkalinephosphatase since protein G has a higher affinity for fallow deerimmunoglobulin than does protein A. Relative to the affinities ofprotein A and protein G for rabbit immunoglobulin (the reference serum),the affinities of protein A and protein G for fallow deer immunoglobulinare 8 and 89% respectively. Fallow deer anti-SIZP titers are uncorrectedfor relative affinity of protein G (Table 5A). None of the does examined2 months or more following the rut and 8-9 months after being immunizedwith the vaccine containing FCA were pregnant, while 96% (192/200)untreated does are pregnant. Pregnancy is determined by examination ofthe reproductive tract for signs of pregnancy or by analyzing blood fromlive captured does for pregnancy-specific protein B (PSPB) byBioTracking, Inc. of Moscow, Id. (Willard et al., “Pregnancy detectionand the effects of age, body weight, and previous reproductiveperformance on pregnancy status and weaning rates of farmed fallow deer(Dama dama). J. Animal Science. 77:32-38 (1999), the disclosure of whichis hereby incorporated by reference). The contrast between the pregnancyrates of immunized and unimmunized does shows clearly that the vaccinecomposition containing FCA is effective in preventing conception. Sincethis is a multiple year study, as many as possible of the does are livecaptured.

TABLE 5A Production of anti-SIZP antibodies by fallow deer immunizedwith the vaccine composition of the invention containing FCA adjuvant.Post-immunization anti-SIZP titer (% of reference serum) Fallow deerTime (months) ID No. 0 1-2 7-10 Controls 99023 0 0 0 99024 0 0 0 99025 00 0 99027 0 0 0 99028 0 0 0 99029 0 0 0 Vaccine with FCA 99026 0 117 ND99025 0 72 ND 2000-06 0 96 ND 2000-08 0 94 ND 99007 0 ND 36 99008 0 ND60 99009 0 ND 94 99016 0 ND 60 99017 0 ND 66 99019 0 ND 133 99020 0 ND56 ND = not determined

Other experiments were performed on white-tailed deer. None of thewhite-tailed deer immunized with the vaccine composition comprising FCAbecame pregnant one year post-immunization. Only one of the white-taileddeer immunized with the vaccine composition comprising alum did notbecome pregnant one year post-immunization. The results for anti-SIZPtiter levels are shown in Table 5B.

TABLE 5B Production of anti-SIZP antibodies by white-tailed deerimmunized with a composition of the present invention. White- tailedAnti-SIZP (% of reference serum) deer ID Post-immunization (months) No.0 2 4 5 8 12 Freund's complete adjuvant (FCA) 19 0 ND ND 85 ND 21 0 NDND 139 ND 33 0 ND ND 125 ND Alum 949 1 12 11 ND 48 27 916 1 6 4 ND 5 2744 3 105 111 ND 123 75 694 0 7 7 ND 5 4 956 0 5 4 ND 2 2 9 0 ND ND 3 NDND 14 0 ND ND 8 ND ND 17 0 ND ND 4 ND ND 27 0 ND ND 4 ND ND ND = notdetermined

Example 6 The Effect of Oil Content on the Production of Anti-SIZPAntibodies

The vaccine composition yields good antibody titers following a singleadministration of an antigen, therefore, unless stated otherwise alltiters reported in the following example results from a singleadministration of the antigen in the vaccine formulation and otherimmunization protocols.

To determine if an aqueous phase is a necessary component of the vaccinecomposition to obtain a good immune response, three groups of rabbits (2or 3 rabbits/group) are immunized with three different preparations ofthe vaccine containing SIZP (50 μg SIZP/rabbit) encapsulated inliposomes that are suspended in saline (0.5 ml) and emulsified inFreund's complete adjuvant (0.5 ml). The proportion of oil phase andwater phase is equal in these preparations (Table 6A).

TABLE 6A Effect of oil content of the vaccine composition on theproduction of anti-SIZP antibodies by rabbits Anti-SIZP titer (% ofreference serum)¹ Oil content Post-immunization (months) (%, v/v) 0 1 23 4  50² 0 120 131 103 24 0 38 38 24 17  50² 0 91 91 54 67 0 46 112 143112  50² 0 54 65 51 ND 0 95 94 81 ND 0 47 49 32 ND 100³ 0 61 75 136 34 014 24 100 95 100⁴ 0 159 149 215 27 0 50 196 244 128 100⁴ 0 11 30 48 29 015 28 40 41 100⁴ 0 54 54 90 91 0 14 2 19 19 0 47 49 67 76 ¹Each linepresents titers of blood samples taken from the same rabbit. ²Liposomescontaining SIZP (50 μg/rabbit) were suspended in saline (0.5 ml) andthis aqueous phase was emulsified in Freund's complete adjuvant (0.5ml). ³Liposomes containing SIZP (50 μg/rabbit) were freeze-dried and theresulting freeze-dried liposomes suspended in Freund's complete adjuvant(0.5 ml). ⁴Liposomes containing SIZP (50 μg/rabbit) are freeze-dried andthe resulting freeze-dried liposomes suspended in Freund's completeadjuvant (0.2 ml). ND = not determined.The vaccine formulated to contain no water is used to immunize fourgroups of rabbits (2 or 3 rabbits/group) with four differentpreparations of the vaccine containing SIZE (50 μg SIZP/rabbit)encapsulated in freeze-dried liposomes suspended in Freund's completeadjuvant (0.2 ml or 0.5 ml). Since Freund's complete adjuvant containsno water, these preparations are water-free and contained only an oilphase. Average anti-SIZP titers 4 months post-immunization are 55+44%(coefficient of variation, cv 80%) for rabbits that are immunized withthe composition containing 50% oil and 59+41% (cv 69%) for rabbits thatare immunized with the vaccine containing 100% oil. There is nodifference in response of female rabbits that received the vaccine with100% oil and female rabbits that are immunized with the vaccinecontaining 50% oil (P=0.87; F (1.45)=0.03; average titers were 71+7% for50% oil and 72+12% for 100% oil). These results indicate that thepresence of an aqueous phase is not necessary for a good immune responseto the vaccine.

To determine if there is a difference in duration of anti-SIZP titers inrabbits that are immunized with the vaccine composition with 50% and100% oil, anti-SIZP titers are measured for 12 months (Table 6B).Anti-SIZP titers during the 12 month post-immunization period aresimilar in rabbits immunized with 50% oil formulation and the rabbitimmunized with 100% oil formulation. To verify the biological effect ofimmunization with SIZP, proven female rabbits immunized with bothformulations of the vaccine are mated with proven males 3 times duringthe 12 month post-immunization period. Reduction in fertility was 80%for the rabbits that are immunized with the vaccine containing 50% oiland the female rabbit that is immunized with the vaccine containing 100%oil produce no offspring indicating that the biological effect ofreduced fertility is similar with both formulations of the vaccine.

TABLE 6B Effect of oil content of the vaccine composition on theduration of anti-SIZP antibodies in rabbits Anti-SIZP titer (% ofreference serum) Rabbit Post-immunization (months) ID No. 0 1 2 3 4 5 67 11 12 50% oil content 3 0 145 112 94 82 45 23 24 20 20 15 0 100 71 6883 19 29 26 18 25 16 0 125 111 122 128 111 74 92 75 63 100% oil content1 0 127 138 ND ND ND 33 51 17 27 ND = not determined.

Since liposomes are composed of material that is lipophilic, storage ofliposomes in oil may lead to their destruction by dissolving theconstituents of liposomes in the oil. To investigate this question,rabbits (2 rabbits in each group) are immunized with the vaccine (100%oil formulation) that is stored for up to 5 months at 5° C. and −20° C.Storage of the vaccine at 5° C. for 5 months reduced anti-SIZP titers ofrabbits by only 28% (Table 6C; P=0.002; F (5.33)=4.9). Storage of thevaccine at −20° C. for 5 months reduced anti-SIZP titers by only 14%(Table 6D; P=<0.001; F (5.35)=23.7). These results indicate that mostliposomes remain intact in oil since immunization of rabbits with asingle injection of SIZP suspended in Freund's complete adjuvant withoutliposome encapsulation results in low titers (Table 6E).

TABLE 6C Effect of storage of the vaccine composition with 100% oilformulation on the production of anti-SIZP antibodies by rabbitsAnti-SIZP titer¹ (% of reference serum) Storage² Post-immmunization(months) (months) 0 1 2 3 4 5 6 Average SE 0 0 60 123 112 163 109 119114 11.2 0 26 108 112 163 131 141 1 0 26 112 183 121 122 100 113 11.9 077 133 142 117 70 153 2 0 63 176 128 70 127 57 98 13.9 0 50 110 100 NDND ND 3 0 23 138 168 104 117 143 94 13.3 0 35 100 113 76 63 42 4 0 17 39104 63 73 100 68 10.1 0 47 136 73 26 45 85 5 0 22 127 69 66 140 99 8211.8 0 27 111 57 54 140 74 ¹The vaccine composition (100% oilformulation) is placed in biobullets purchased from Ballistivet ™ andsurgically implanted intramuscularly into rabbits. ²Biobullets arestored at 5° C.

TABLE 6D Effect of storage of the vaccine composition with 100% oilformulation on the production of anti-SIZP antibodies by rabbitsAnti-SIZP titer¹ (% of reference serum) Storage² Post-immunization(months) (months) 0 1 2 3 4 5 6 Average SE 0 0 60 123 112 163 109 119114 11.2 1 26 108 112 163 131 141 1 0 9 13 13 41 19 60 27 6.7 0 6 16 1473 33 ND 2 0 9 14 23 70 136 116 51 12.7 0 6 30 19 41 95 57 3 0 13 23 127100 73 88 57 11.1 0 13 18 90 61 58 30 4 0 13 68 50 71 80 122 85 14.0 013 114 134 81 100 179 5 0 9 114 75 87 146 92 98 13.7 1 22 108 116 109179 125 ¹The vaccine composition (100% oil formulation) is placed inbiobullets purchased from Ballistivet ™ and surgically implantedintramuscularly into rabbits. ²Biobullets are stored at −20° C.

TABLE 6E Production of anti-SIZP antibodies by rabbits immunized with asingle administration of SIZP without encapsulation of SIZP in liposomesAnti-SIZP titer (% of reference serum)¹ Post-immunization (months)Rabbit ID No. 0 1 2 3 4 1 0 43 19 9 2 2 0 27 7 4 1 ¹Rabbits areimmunized with a single administration of SIZP (50 μg/rabbit) suspendedin Freund's Complete Adjuvant.

To determine if the vaccine formulated to contain no aqueous phase wouldresult in a good response in another mammalian species, grey seals(Halichoerus grypus) are immunized with the vaccine containing eitherequal oil and aqueous phases, only an aqueous phase, or only an oilphase (Table 6F). There was no difference in anti-SIZP titers in thevaccine that contained equal oil and aqueous phases or only oil butadministration of the vaccine that contained all ingredients except oil,resulted in significantly lower titers.

TABLE 6F Effect of oil content of the vaccine composition on theproduction of anti-SIZP antibodies by grey seals. Anti-SIZP titer (% ofreference serum)¹ Oil content Post-immunization (months) (%, v/v) 0 1 23 4  0² 0 5 4 1 1 0 1 2 1 1  50³ 0 7 9 145 107 0 41 52 82 38 100⁴ 0 2028 79 60 0 3 30 90 50 ¹Each line presents titers of blood samples takenfrom the same grey seal. ²Liposomes containing SIZP (100 μg/grey seal)and heat killed Mycobacterium tuberculosis (2 mg/grey seal), the activeingredient in Freund's complete adjuvant as supplied by Sigma ChemicalCo. and used in all studies reported herein are suspended in saline (0.5ml). ³Liposomes containing SIZP (100 μg/grey seal) are suspended insaline (0.5 ml) and this aqueous phase is emulsified in Freund'scomplete adjuvant (0.5 ml). ⁴Liposomes containing SIZP (100 μg/greyseal) are freeze-dried and the resulting freeze-dried liposomessuspended in Freund's complete adjuvant (0.5 ml).

Example 7 Use of Archaebacterial Lipids in Liposomes

Liposomes are completely closed lipid membranes that can be made from avariety of lipid materials. In this example, liposomes made usingarchaebacterial lipids are compared to liposomes made using soybeanlecithin for their ability to stimulate antibody production by rabbits(Table 7). Liposomes made with soybean lecithin result in betterproduction of anti-SIZP antibodies than liposomes made witharchaebacterial lipids.

TABLE 7 Production of anti-SIZP antibodies by rabbits immunized withliposomes prepared with archaebacterial lipids or soybean lecithin.Anti-SIZP titer (% of reference serum) Post immunization (months) ID No.Type of lipid 0 1 2 3 4 5 112 Soybean lecithin 0 143 195 137 197 98 115Soybean lecithin 0 200 198 157 179 106 117 Archaebacterial lipids 0 4630 37 13 ND 118 Archaebacterial lipids 0 7 4 8 2 ND ND = not determined.

Example 8 Immunization Against Streptokinase

The vaccine composition is unique in producing high titers of anti-SIZPantibodies that are long-lasting following a single administration. Todetermine if the vaccine composition would produce high antibody titerswith other antigens, rabbits are immunized with streptokinase.

Streptokinase is an exoprotein produced by pathogenic strains of theStreptococci family of bacteria. As an activator of vascularfibrinolysis its therapeutic usefulness has been appreciated for manyyears in the treatment of myocardial infarction. Streptokinase unfoldsin a non-cooperative manner. Therefore, the protein can assume a numberof partially folded states that contain some regions that appear to benative and others that are unfolded. Three domains of differentstability exist that are independent of other regions of the protein(Teuten et al., 1993, Biochem. J. 290:313-319). Native streptokinasecontains immunodominant epitopes in the C-terminal region (Torrens etal., 1999, Immunology Letters 70:213-218). The C-terminal region isrelatively unstructured (Parrado et al., 1996, Protein Sci 5:693-704)therefore heat treatment cannot alter the structure since it wasunstructured before heat treatment. The thermal stability of domain C issignificantly increased by its isolation from the rest of the chain(Connejero-Lara et al., 1996, Protein Sci 5:2583-2591). Loss of theC-terminal region results in a less immunogenic protein but does exposeimmunogenic epitopes hidden in the native molecule. In our studies, theC-terminal region was present in native and heat-treated streptokinase,therefore, as the immunodominant region of the protein, it woulddetermine the response of the rabbits. If the C-terminal region retainedthe same epitopes following heat treatment as found in the native state,one would not expect to find a difference in binding ofanti-streptokinase antibodies to native and heat-treated streptokinase.These are exactly the observations found (Table 8). We have proposedthat delivery of denatured proteins using a vaccine composition of thepresent invention favours the production of antibodies directed againstnative epitopes. This is supported by the studies of alcoholdehydrogenase in Example 2. The results with streptokinase areconsistent with this proposal since heat treatment: would not alter thestructure of the immunodominant region and the prediction follows thatthere would be no difference in the immune response of rabbits beingimmunized with native and heat treated streptokinase regardless of thedelivery system employed. These are precisely our observations (Table8).

TABLE 8 Epitope mapping of rabbit anti-streptokinase sera from rabbitsimmunized with native and heat-treated streptokinase (100° C. for 10minutes in 5% mercaptoethanol) using conventional immunizationprotocols¹ or the method of the present invention². Titer (% ofreference serum)³ Native Heat-treated Immunization streptokinasestreptokinase Rabbit Post-immunization (months) ID Antigen Delivery 0 12 0 1 2 21 Native Invention 0 100 122 0 98 122 24 Native Invention 0 8298 0 53 108 25 Native Conventional 0 10 89 0 8 100 20 NativeConventional 0 9 94 0 3 92 23 Heat- Invention 0 10 34 0 15 31 treated 28Heat- Invention 0 25 99 0 24 94 treated 27 Heat- Conventional 0 9 107 07 94 treated 30 Heat- Conventional 0 10 100 0 8 94 treated ¹Rabbits wereimmunized with 75 μg streptokinase in Freund's complete adjuvantfollowed by one booster injection one month later with 75 μgstreptokinase in Freund's incomplete adjuvant. ²Rabbits were immunizedwith a single injection of 75 μg streptokinase in a vaccine of thepresent invention. ³Titers were measured with both native andheat-treated streptokinase.

It is evident from the results that a single injection of streptokinaseusing a vaccine of the present invention produced anti-streptokinasetiters similar to titers obtained by the conventional primary andbooster injection protocols. Also, regardless of the immunizationprotocol used, that is the present invention or conventional, theantibodies produced bound to native and heat-treated streptokinaseequally well.

Example 9 Use of an Edible Vegetable Oil

A vaccine composition was formulated in accordance with this inventionusing Canola oil in place of mineral oil. The results are shown in Table9. The results indicate that vaccines formulated with Canola oil produceanti-SIZP antibodies in rabbits, therefore, Canola oil is useful.However, the titer levels are not as high as with mineral oil.

TABLE 9 Effect of Canola oil on production of anti-SIZP antibodies inrabbits Anti-SIZP titer (% of reference serum) Post-immunization(months) Rabbit ID Vaccine formulation 0 1 2 3 4 5 6 7 4 Alum/mineraloil 0 111 123 105 124  95 125 157 14 0 231 132 205 178 279 258 251 9FCA/mineral oil¹ 0 162 264 310 ND ND ND ND 26 0 351 166 112 ND ND ND ND34 FCA/Canola oil² 0 27 24 37 ND ND ND ND 36 0 18 24 36 ND ND ND ND¹Freund's complete adjuvant (FCA) was obtained from a commercial source(Sigma) and was formulated with mineral oil. ²FCA/Canola oil containedthe same quantity of Mycobacterium heat-killed cells as present in FCAbut the mineral oil component of FCA was replaced with edible Canolaoil. ND = not determined

Example 10 Immunization Against Hepatitis B

A hepatitis B vaccine was formulated in accordance with the presentinvention using 5 micrograms hepatitis B surface antigen (RecombivaxHB™, a recombinant hepatitis B antigen) containing alum adjuvantencapsulated in liposomes containing soybean lecithin (0.05 g) andcholesterol (0.005 g) suspended in saline (0.25 ml) then emulsified inlow viscosity mineral oil (0.225 ml) and mannide oleate (0.025 ml). Aconventional hepatitis B vaccine using 5 micrograms hepatitis B surfaceantigen (Recombivax HB™) containing alum adjuvant in a volume of 0.5 mlaqueous medium as recommended by the manufacturer was also administered.Eight rabbits were immunized with the vaccine prepared in accordancewith the present invention and eight rabbits were immunized with theconventional vaccine. Results are shown in Table 10.

It is evident from Table 10 that the vaccine prepared in accordance withthe present invention results in about 6 times more antibody 1 monthpost-immunization than conventional delivery of hepatitis B surfaceantigen.

TABLE 10 Production of anti-HepB antibodies by rabbits immunized with acommercial HepB vaccine or with a HepB vaccine formulated in accordancewith the present invention Anti-HepB titer (mlU/ml)¹ Post-immunization(months) Rabbit ID 0 1 Commercial vaccine 96 0 736 101 0 1237 97 0 488100 0 1877 99 0 6251 103 0 8384 98 0 688 102 0 1568 Average 0 2654Vaccine of the invention 93 0 32,341 95 0 3371 88 0 5717 81 0 23,808 830 9344 84 0 17,856 79 0 9344 85 0 21,675 Average 0 15,432 ¹Antibodytiters were measured using the enzyme immunoassay for the detection ofantibody to hepatitis B surface antigen (anti-HBs) distributed byDiaSorin Inc., Stillwater, MN, USA.

Example 11 Effect of Formulating Vaccines with Alum Adjuvant Inside andOutside of Liposomes

Vaccines were prepared as follows:

Group SIZP antigen Alum Medium 1 inside liposome inside liposome saline2 inside liposome outside liposome saline 3 inside liposome insideliposome oil 4 inside liposome outside liposome oil 5 control - noliposomes oil 6 control - no liposomes saline

Groups 1-4 were prepared with 100 μg SIZP encapsulated in liposomesformed with 0.1 g soybean lecithin and 0.01 g cholesterol. The liposomesin Groups 1 and 3 also contained 100 μl ImjectAlum™. In Groups 2 and 4,100 μl ImjectAlue™ was placed outside the liposomes. In Groups 1 and 2,the liposomes were suspended in 0.25 ml saline and this suspensionemulsified in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate. In Groups 3 and 4, the liposomes were freeze dried thensuspended in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate and this suspension emulsified in 0.25 ml saline. In Group 5, 100μg SIZP and 100 μl ImjectAlum™ were freeze dried, then suspended in0.225 ml low viscosity mineral oil and 0.025 ml mannide oleate andemulsified in 0.25 ml saline. In Group 6, 100 μg SIZP and 100 μlImjectAlum™ were freeze dried, then suspended in 0.25 ml saline andemulsified in 0.225 ml low viscosity mineral oil and 0.025 ml mannideoleate. Rabbits were immunized with the six groups of vaccines and theresults are shown in Table 11.

TABLE 11 Production of anti-SIZP antibodies by rabbits immunized withfour formulations of a vaccine of the present invention containing alumadjuvant (Groups 1-4) and two control formulations containing alumadjuvant (Groups 5-6) Anti-SIZP titer Standard (% reference serum)Average titer Error of Post-immunization Post-immunization averagemonths months titer Group Rabbit ID 0 1 2 3 0 1 2 3 1 2 3 1 49 2 107 176183 0 135 203 236 22 28 37 1 76 0 134 105 124 1 71 0 70 249 331 1 82 0182 236 268 1 78 0 182 249 274 2 73 0 373 273 304 0 287 207 258 32 24 152 42 0 328 199 281 2 62 0 259 194 244 2 77 0 182 131 235 2 74 0 295 238225 3 63 0 363 135 113 0 300 171 160 32 26 34 3 67 0 286 175 140 3 70 0332 241 261 3 80 0 218 131 125 4 48 0 383 210 113 0 200 138 108 49 20 124 64 0 129 109 121 4 61 0 125 98 63 4 60 0 148 140 115 4 66 0 215 134130 5 45 0 21 133 120 0 26 96 123 8 29 33 5 65 0 6 26 42 5 69 0 49 110121 5 72 0 12 36 89 5 50 0 40 176 242 6 68 0 28 31 22 0 16 22 18 4 3 1 675 0 9 16 16 6 46 0 18 23 17 6 43 0 10 19 16

Example 12 Effect of Formulating Vaccines with Heat Killed Mycobacteriumtuberculosis Adjuvant Inside and Outside of Liposomes

Vaccines were prepared as follows:

Heat-killed Group SIZP antigen M. tuberculosis Medium 1 inside liposomeinside liposome saline 2 inside liposome outside liposome saline 3inside liposome inside liposome oil 4 inside liposome outside liposomeoil

Groups 1-4 were prepared with 100 μg SIZP encapsulated in liposomesformed with 0.1 g soybean lecithin and 0.01 g cholesterol. The liposomesin Groups 1 and 3 also contained 200 μg heat killed M. tuberculosis. InGroups 2 and 4, 200 μg heat killed M. tuberculosis was placed outsidethe liposomes. In Groups 1 and 2, the liposomes were suspended in 0.2 mlsaline and this suspension emulsified in 0.18 ml low viscosity mineraloil and 0.02 ml mannide oleate. In Groups 3 and 4, the liposomes werefreeze dried then suspended in 0.18 ml low viscosity mineral oil and0.02 ml mannide oleate and this suspension emulsified in 0.2 ml saline.Rabbits were immunized with the four groups of vaccines and the resultsare shown in Table 12.

TABLE 12 Production of anti-SIZP antibodies by rabbits immunized withfour formulations of a vaccine of the present invention containing heatkilled M. tuberculosis Anti-SIZP titer (% Standard reference serum)Average titer Error of Post-immunization Post-immunization averageRabbit months months titer Group ID 0 1 2 3 0 1 2 3 1 2 3 1 33 0 245 236259 0 318 262 437 51 18 126 1 29 0 390 288 614 2 32 0 544 515 633 0 576532 638 22 12 3 2 22 0 608 549 642 3 9 0 162 264 310 0 293 599 508 93237 140 3 13 0 424 933 707 4 16 0 454 276 532 0 403 221 322 36 39 149 426 0 351 166 112

Example 13 Immunization of Rabbits with Native and Denatured YeastAlcohol Dehydrogenase (ADH) Together with Alum Adjuvant

Vaccines of the present invention were formulated containing 100 μg ofnative or denatured ADH together with 100 μl ImjectAlum™ encapsulated inliposomes formed with 0.1 g soybean lecithin and 0.01 g cholesterol. Theliposomes were suspended in 0.25 ml saline and the suspension emulsifiedin 0.225 ml low viscosity mineral oil and 0.025 ml mannide oleate.

Conventional vaccines were formulated containing 100 μg of native ordenatured ADH together with 100 μl ImjectAlum™ and suspended in 0.5 mlsaline.

Denatured ADH was prepared by heating ADH to 100° C. for 30 minutes andtreating with 10% mercaptoethanol for 30 minutes at room temperature tocleave disulfide bonds. Mercaptoethanol was removed by dialysis for 12hours and denatured ADH was recovered by freeze drying.

Results comparing the vaccines of the present invention to theconventional vaccines are shown in Table 13.

TABLE 13 Production of anti-ADH antibodies by rabbits immunized withnative and denatured ADH using alum as adjuvant Anti-ADH titer (%reference serum) Post-immunization (months) Rabbit Delivery Native ADHDenatured ADH ID System 0 1 2 3 0 1 2 3 Native ADH 54 invention 0 24 4266 0 8 4 4 57 0 100 143 146 0 29 3 6 55 0 91 85 111 0 26 3 5 40conventional 0 1 1 2 0 1 0 1 44 0 1 1 1 0 1 0 1 47 0 5 4 8 0 3 1 1Denatured ADH 53 invention 0 1 2 5 0 1 1 2 58 0 1 1 2 0 1 0.5 1 52 0 1 415 0 1 0.5 1 51 conventional 0 2 4 4 0 1 0.5 2 59 0 1 1 4 0 1 0.5 0 56 02 2 2 0 1 0.5 0

The results show that delivery of native or denatured ADH using aformulation of the present invention results in an increased productionof anti-ADH antibodies compared to the production of anti-ADH antibodiesby rabbits immunized against native or denatured ADH using conventionalmethods. Furthermore, rabbits immunized with denatured ADH produced moreantibodies directed against native ADH when a formulation of the presentinvention is used rather than when denatured ADH is delivered byconventional means with no booster injections.

Example 14 Epitope Mapping

Epitope mapping experiments to demonstrate that vaccines of the presentinvention produce antibodies having different binding specificity for anantigen than achieved by conventional immunization protocols of primaryand secondary booster injections. Fragments of the ZP antigen werespecifically used but it is expected that other antigens will behave ina similar manner.

Conventional immunization of grey and harp seals with a primaryinjection and two booster injections results in low anti-SIZP antibodytiters that peak two months post-immunization in both grey and harpseals. In contrast, immunization with a vaccine formulated in accordancewith the present invention produces anti-SIZP antibody titers thatpersist for at least 24 months in grey seals and 5-6 months in harpseals, with one exception. Titers in harp seals reach a plateau thatpersist for 6-10 months post-immunization. Therefore, a vaccineformulated in accordance with the present invention induces highanti-SIZP titers with long duration compared to conventionalimmunization protocols using primary and booster injections.

The polypeptide fragments of ZPB and ZPC that were used in epitopemapping to demonstrate that anti-SIZP antibodies produced followingconventional immunization protocols have a different binding specificitythan anti-SIZP antibodies produced following immunization with a vaccineof the present invention are shown in FIG. 1. The fragments ZPB1, ZPB2,ZPC1 and ZPC2 are short length polypeptides and do not have thethree-dimensional structures of full length ZPB and ZPC. In FIG. 1, thefull-length unprocessed polypeptides are shown above the two ZPB and ZPCfragments. The secretory signal peptides that are cleaved in the nativeproteins are shaded in black.

Anti-SIZP grey seal antibodies produced following conventionalimmunization (a primary and two booster injections using FCA adjuvant)have a high affinity for the ZPB1, ZPB2, ZPC1 and ZPC2 fragments (Table14A, seal ID 1). In contrast, grey seals immunized with a vaccineformulated in accordance with the present invention (Table 14A, seal ID76 and 96) produce antibodies that have a low affinity for fragmentsZPB2, ZPC1 and ZPC2 one year post-immunization and low affinity for allfour fragments three years post-immunization. The four fragmentstogether account for 80% of the protein found in SIZP.

TABLE 14A Epitope mapping of grey seal anti-SIZP antibodies usingrecombinant fragments of ZPB and ZPC produced in E. coli Post- Sealimmunization Binding relative to SIZP (%) ID (months) ZPB1 ZPB2 ZPC1ZPC2 Total 1 3 30 44 59 41 174 1 4 71 70 83 63 287 76 12 54 25 8 12 9976 36 18 9 13 11 51 96 12 47 18 15 10 90 96 36 10 8 15 10 43

A temporal study of the binding specificity of antibodies produced bygrey seals immunized with a vaccine formulated in accordance with thepresent invention indicated that antibodies produced earlypost-immunization (<7 months) bind to epitopes found predominantly onthe ZPB1 fragment. Antibodies produced late post-immunization (>7months) have lower affinity for ZPB1 and the other three fragments.ZPB1, ZPB2, ZPC1 and ZPC2 are low molecular weight and are notglycosylated. These fragments have less three-dimensional structure thanfull-length ZPB and ZPC because of their low molecular weight.Therefore, antibodies that bind to SIZP but not ZPB1, ZPB2, ZPC1 or ZPC2must either be recognizing three-dimensional structures found only onfull-length ZPB and ZPC or the carbohydrate covalently linked to theseproteins. Since the total amount of antibody bound to the fragmentsearly post-immunization exceeds or is equivalent to the amount ofantibody binding to ZPB and ZPC, carbohydrate-recognizing antibody musthave a minor role. This implies that 3-D structures determine thedifference in binding to the fragments as opposed to SIZP. A survey ofnine other grey seals immunized with SIZP in a vaccine of the presentinvention indicates similar reduction to antibodies produced 5 months ormore post-immunization.

In another experiment, three of four rabbits immunized with a vaccine ofthe present invention produced antibodies with a higher affinity forepitopes in ZPB1 than in the other three fragments. Only 20-40% of theantibodies produced by all four rabbits bound to epitopes found in thefour ZP fragments. Therefore, 60-80% of the anti-SIZP antibodiesproduced by rabbits immunized with a vaccine of the present inventionbound only to epitopes found in full length ZPB and ZPC. Therefore,60-80% of antibodies produced in rabbits immunized with a vaccine of thepresent invention recognize epitopes related to native 3-D structures.

In yet another experiment, immunization of harp seals (156 and 162) byconventional protocols of a primary injection using FCA adjuvantfollowed by booster injections with FIA adjuvant produced antibodiesearly post-immunization that bound to epitopes found in ZPB1, ZPB2 andZPC2 (harp seal 156) or all four fragments (harp seal 162) as well as inSIZP (Table 14B). In contrast, immunization of harp seal 151 with avaccine formulated in accordance with the present invention producedantibodies early post-immunization (<5 months) that bound to epitopesfound in all four ZP fragments but antibodies produced latepost-immunization (>7 months) bound to epitopes found only on fulllength ZPB and ZPC (Table 14B). Only 30-40% of the antibodies producedby immunization of harp seal 153 with a vaccine of the present inventionbound well to epitopes on the four ZP fragments, implying that 60-70% ofthe antibodies produced by harp seal 153 during the 7 monthpost-immunization period bound only to epitopes found in full length ZPBand ZPC. These epitopes must be related to structures found only in fulllength ZPB and ZPC implying 3-D structures. Immunization of hooded seal1 with a vaccine of the present invention produced antibodies with asimilar temporal sequence of specificity as harp seal 151.

TABLE 14B Epitope mapping of harp and hooded seal anti-SIZP antibodiesusing recombinant fragments of ZPB and ZPC produced in E. coli Post-immunization Binding relative to SIZP (%) Seal ID (months) ZPB1 ZPB2ZPC1 ZPC2 Total Harp 156 2 9 8 7 7 31 3 30 19 10 24 83 4 36 38 2 34 110Harp 162 2 8 10 11 11 40 3 33 26 19 24 102 Harp 151 1 40 33 36 33 142 341 28 32 21 122 5 21 27 35 34 117 6 50 26 30 44 150 7 8 6 9 8 31 9 4 1732 10 63 Harp 153 2 12 6 11 9 38 3 16 12 9 12 49 4 6 5 7 3 21 5 13 8 1211 44 6 12 9 11 12 44 7 9 6 0 7 22 Hooded 1 2 30 22 26 27 105 3 33 25 2830 116 4 37 32 38 34 141 5 31 24 29 31 115 7 15 12 12 11 50 8 12 13 11 844

1. A composition for use as a vaccine, comprising: (a) a carriercomprising a continuous phase of a hydrophobic substance; (b) liposomes;(c) an antigen; and, (d) an adjuvant.
 2. The composition of claim 1,wherein the carrier is an oil or a water-in-oil emulsion.
 3. Thecomposition of claim 2, wherein the oil is mineral oil, a vegetable oilor a nut oil.
 4. The composition of claim 3, wherein the adjuvant isalum, another compound of aluminum or TiterMax.
 5. The composition ofclaim 3, wherein the antigen is a suitable native, non-native,recombinant or denatured protein or peptide, or a fragment thereof;and/or wherein the antigen is a viral, bacterial, protozoal or mammalianantigen.
 6. The composition of claim 5, wherein the antigen is capableof eliciting an antibody that recognizes a native epitope, such as anative mammalian epitope.
 7. A method for potentiating an immuneresponse in an animal, which method comprises administering to theanimal an effective amount of a vaccine composition comprising: (a) acarrier comprising a continuous phase of a hydrophobic substance; (b)liposomes; (c) an antigen; and, (d) an adjuvant.
 8. The method of claim7, wherein the carrier is an oil or a water-in-oil emulsion.
 9. Themethod of claim 8, wherein the oil is mineral oil, a vegetable oil or anut oil.
 10. The method of claim 9, wherein the adjuvant is alum,another compound of aluminum or TiterMax.
 11. The method of claim 8,wherein the antigen is capable of eliciting an antibody that recognizesa native epitope, such as a native mammalian epitope.
 12. A method ofpreparing a vaccine composition comprising the steps of: (a)encapsulating an antigen or an antigen/adjuvant complex in liposomes toform liposome-encapsulated antigen; (b) mixing the liposome-encapsulatedantigen with a carrier comprising a continuous phase of a hydrophobicsubstance; and, (c) adding an adjuvant if an antigen/adjuvant complex isnot used in part (a).
 13. The method of claim 12, wherein an antigenwithout adjuvant is encapsulated in the liposomes before adding theadjuvant and the liposome-encapsulated antigen is freeze-dried afteradding the adjuvant to form a freeze-dried liposome-encapsulated antigenwith external adjuvant.
 14. The method of claim 13, wherein: theadjuvant is added to pyrogen-free water before the adjuvant is added tothe liposome-encapsulated antigen; and/or the freeze-driedliposome-encapsulated antigen with external adjuvant is mixed with thecarrier, and wherein an aqueous medium is mixed with the carrier to forman emulsion of water-in-the hydrophobic substance.
 15. The method ofclaim 12, wherein the liposome-encapsulated antigen comprises anantigen/adjuvant complex, and wherein the liposome-encapsulated antigenis freeze-dried before it is mixed with the carrier, and wherein anaqueous medium is mixed with the carrier to form an emulsion ofwater-in-the hydrophobic substance.
 16. The method of claim 12, wherein:(i) the liposome-encapsulated antigen is mixed with an aqueous mediumbefore being mixed with the carrier; (ii) the adjuvant is added to thecarrier before the carrier is mixed with the liposome-encapsulatedantigen; and, (iii) the carrier is mixed with the liposome-encapsulatedantigen to form an emulsion of water-in-the hydrophobic substance. 17.The method of claim 12, wherein: (i) the liposome-encapsulated antigencomprises an antigen/adjuvant complex; (ii) the liposome-encapsulatedantigen is mixed with an aqueous medium before being mixed with thecarrier; and, (iii) the liposome-encapsulated antigen is mixed with thecarrier to form an emulsion of water-in-the hydrophobic substance. 18.The method of claim 12, wherein the carrier is an oil or a water-in-oilemulsion.
 19. The method of claim 18, wherein the oil is mineral oil, avegetable oil or a nut oil.
 20. The method of claim 12, wherein thecarrier is adjuvant is alum, another compound of aluminum or TiterMax.21-43. (canceled)